Summary: This proposal is responsive to RFA: Juvenile Protective Factors and Their Effects on Aging (R01) Given their broad regulatory roles, knowledge of microRNA (miRNA) biology is essential to a comprehensive understanding of aging. The mode of miRNA-mediated regulation is special in that hundreds of genes can be targeted by a single miRNA while multiple miRNAs can target the same gene. This framework commends miRNAs as key modulators of complex phenotypes by integrating multiple biological inputs and outputs. The multiplicity of pathways and processes that are united by miRNAs leads to highly complex network diagrams to better understand the biology of aging and for applications such as therapeutics development. Since miRNAs have therapeutic uses, e.g. application of exogenous miRNAs to alleviate over-expression of detrimental aging-associated genes, our hope is manipulation of miRNA levels would be a potential therapeutic tool in diseases of aging. My work in developmental biology inspired my move towards understanding the role of microRNAs (miRNAs) in stem cells and cancer in more complex organisms, and towards understanding roles for miRNAs in aging. We identified the first miRNA (gerontomiR) to be implicated in the aging process, lin-4. Knockdown of lin-4 in Caenorhabditis elegans decreased longevity of the worms, whereas over-expression of lin-4 increased longevity. Additionally, overexpression of miR-71 and miR-246 in C. elegans increased longevity. Interestingly, most of these gerontomiRs show peak expression during juvenile/larval and early adult phases of the lifecycle, and yet are important for normal aging in adults. For example, lin-4 and let-7 play key roles in stem cell developmental timing in larvae while miR-71 and miR-246 are required for stress response in larvae, yet all are down-regulated in the adult and are required to promote healthy aging. Additionally, increased expression levels of mir-71 and miR-246 in early adulthood of individual animals is associated with longer individual lifespan. In this new work will identify additional gerontomiRs and test the hypothesis that heterochronic expression of these gerontomiRs in the adult is capable of extending lifespan in C. elegans. Recently, we and other have identified various miRNAs that are up- or down-regulated during mammalian aging, by comparing their liver or brain-specific expression in younger and older mice. We have also begun to identify miRNAs that could be useful biomarkers of aging in humans. Given the possibility that miRNAs may regulate processes involved in human aging, here we will also examine the aging pathways that human homologues of the gerontomiRs identified in Aim 1 may function in and test if miRNAs regulate cell senescence of human cells and potentially, mouse models.